Pyruvate dehydrogenase kinase

Phosphorylation sites

PDK can phosphorylate a serine residue on pyruvate dehydrogenase at three possible sites. Some evidence has shown that phosphorylation at site 1 will nearly completely deactivate the enzyme while phosphorylation at sites 2 and 3 had only a small contribution to complex inactivation. Therefore, it is phosphorylation at site 1 that is responsible for pyruvate dehydrogenase deactivation.

Isozymes

There are four known isozymes of PDK in humans:

• PDK1
• PDK2
• PDK3
• PDK4

The primary sequencing between the four isozymes are conserved with 70% identity. The greatest differences occur near the N-terminus.

PDK1 is the largest of the four with 436 residues while PDK2, PDK3 and PDK4 have 407, 406, and 411 residues respectively. The isozymes have different activity and phosphorylation rates at each site. At site 1 in order from fastest to slowest, PDK2 PDK4 ≈ PDK1 PDK3. For site 2, PDK3 PDK4 PDK2 PDK1. Only PDK1 can phosphorylate site 3. However, it has been shown that these activities are sensitive to slight changes in pH so the microenvironment of the PDK isozymes may change the reaction rates.

Isozyme abundance has also been shown to be tissue specific. PDK1 is ample in heart cells. PDK3 is most abundant in testis. PDK2 is present in most tissues but low in spleen and lung cells. PDK4 is predominantly found in skeletal muscle and heart tissues.

Mechanism

Pyruvate dehydrogenase is deactivated when phosphorylated by PDK. Normally, the active site of pyruvate dehydrogenase is in a stabilized and ordered conformation supported by a network of hydrogen bonds. However, phosphorylation by PDK at site 1 causes steric clashes with another nearby serine residue due to both the increased size and negative charges associated with the phosphorylated residue. This disrupts the hydrogen bond network and disorders the conformation of two phosphorylation loops. These loops prevent the reductive acetylation step, thus halting overall activity of the enzyme. The conformational changes and mechanism of deactivation for phosphorylation at sites 2 and 3 are not known at this time.

Regulation

Pyruvate dehydrogenase kinase is activated by ATP, NADH and acetyl-CoA. It is inhibited by ADP, NAD+, CoA-SH and pyruvate.

Each isozyme responds to each of these factors slightly differently. NADH stimulates PDK1 activity by 20% and PDK2 activity by 30%. NADH with acetyl-CoA increases activity in these enzymes by 200% and 300% respectively. In similar conditions, PDK3 is unresponsive to NADH and inhibited by NADH with acetyl-CoA. PDK4 has a 200% activity increase with NADH, but adding acetyl-CoA does not increase activity further.

Disease relevance

PDK isoforms are elevated in obesity, diabetes, heart failure, and cancer. Some studies have shown that cells that lack insulin (or are insensitive to insulin) overexpress PDK4. As a result, the pyruvate formed from glycolysis cannot be oxidized which leads to hyperglycaemia due to the fact that glucose in the blood cannot be used efficiently. Therefore, several drugs target PDK4 hoping to treat type II diabetes.

PDK1 has shown to have increased activity in hypoxic cancer cells due to the presence of HIF-1. PDK1 shunts pyruvate away from the citric acid cycle and keeps the hypoxic cell alive. Therefore, PDK1 inhibition has been suggested as an antitumor therapy since PDK1 prevents apoptosis in these cancerous cells. Similarly, PDK3 has been shown to be overexpressed in colon cancer cell lines. Three proposed inhibitors are AZD7545 and dichloroacetate which both bind to PDK1, and Radicicol which binds to PDK3.

Mutations in the PDK3 gene are a rare cause of X-linked Charcot-Marie-Tooth disease (CMTX6).

In dogs, specifically Doberman Pinschers, a mutation in the PDK4 gene is associated with dilated cardiomyopathy (DCM).

References

References

1. (June 1978). "Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart". Biochemistry.
2. (November 1994). "Molecular cloning of the p45 subunit of pyruvate dehydrogenase kinase". The Journal of Biological Chemistry.
3. (October 2001). "Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase". The Journal of Biological Chemistry.
4. (August 2001). "Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites". The Biochemical Journal.
5. (January 1998). "Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex". The Biochemical Journal.
6. (February 2001). "Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites". The Journal of Biological Chemistry.
7. (December 2008). "Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops". Structure.
8. (August 1974). "Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase". Biochemical and Biophysical Research Communications.
9. (May 2003). "Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs". American Journal of Physiology. Endocrinology and Metabolism.
10. (2018). "Role of the Pyruvate Dehydrogenase Complex in Metabolic Remodeling: Differential Pyruvate Dehydrogenase Complex Functions in Metabolism". Diabetes Medical Journal.
11. (October 1998). "Insulin downregulates pyruvate dehydrogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resistant subjects". Molecular Genetics and Metabolism.
12. (December 2003). "Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation". Biochemical Society Transactions.
13. (March 2006). "HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia". Cell Metabolism.
14. (January 2007). "A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth". Cancer Cell.
15. (September 2011). "Overexpression of pyruvate dehydrogenase kinase 3 increases drug resistance and early recurrence in colon cancer". The American Journal of Pathology.
16. (August 2007). "Distinct structural mechanisms for inhibition of pyruvate dehydrogenase kinase isoforms by AZD7545, dichloroacetate, and radicicol". Structure.
17. {{OMIM. 300905. Charcot-Marie-tooth disease, X-linked dominant, 6; CMTX6
18. (April 2013). "A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene". Human Molecular Genetics.
19. (March 2020). "Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts". Scientific Reports.
20. (2017). "PDK4 Deficiency Induces Intrinsic Apoptosis in Response to Starvation in Fibroblasts from Doberman Pinschers with Dilated Cardiomyopathy". BioResearch Open Access.
21. (August 2012). "A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher". Human Genetics.